Abstract：Rice stripe virus (RSV) is a type member of Tenuivirus, inducing rice (Oryza sativa) stripe disease in East Asia such as Korea, Japan and China, causing large yield loss in rice production. In the present study, yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) technique were used to detect the self-interaction sites of the RSV coat protein (CP). A series of CP deleted mutants were constructed and transformed into yeast strain Y2H Gold, observing the growth of yeast transformants on quadruple drop out SD/-Leu-Trp-His-Ade/X-α-gal agar plates. It was revealed that C terminal domain from residue 307~318 played an essential role in the interaction, deleting or damaging of which abolished the self-interaction. Site-directed mutants were generated to investigate the specific sites of CP that involved in the interaction. It revealed that the L308, V309, F312 and F313 amino acids were critical for CP self-interaction, which were highly conserved in Tenuivirus. BiFC assay was performed to confirm these results. Agrobacterium tumefaciens that carried the CP deleted mutants or site-directed mutants were infiltrated into the epidermal leaves of Nicotiana benthamiana. Confocal microscope was used to observe the yellow fluorescent protein (YFP) 48 h post inoculation (hpi). The CPΔ307~322, CPL308A, CPV309A, CPF312A and CPF313A had no signal, indicating there was no self-interaction, while CPΔ319~322 showed YFP similar to the full-length CP mainly aggregated in cytoplasm, consistent with results in Y2H. The cellular localization of these CP mutants was different from that of the full length wild-type CP. The site-directed mutants that had lost the ability of self-interaction showed diffused localization in the plasma membrane, while a substantial proportion of the full length CP gathered in the cytoplasm, forming numerous discrete aggregates, suggesting that the CP self-interaction and the subcellular localization in plant were interdependent processes. It was presumed that the forms of dimmer or higher multimer of CP might involve in different processes from the individual protein. These results may be useful in understanding the important role of CP in the process of RSV infection.
